Streptomyces silvae sp. nov., isolated from forest soil

Abstract

A bacterial strain, named For3^T, was isolated from forest soil sampled in Champenoux, France. Based on its 16S rRNA gene sequence, the strain was affiliated to the family Streptomycetaceae and, more specifically, to the genus Streptomyces . The strain had 99.93% 16S rRNA gene sequence similarity to its closest relative strains Streptomyces pratensis ATCC 33331^T, Streptomyces anulatus ATCC 27416^T, Streptomyces setonii NRRL ISP-5322^T and Kitasatospora papulosa NRRL B-16504^T. The phylogenomic tree using the genome blast distance phylogeny method showed that the closest relative strain was Streptomyces atroolivaceus NRRL ISP-5137^T and that For3^T represents a new branch among the Streptomyces . Genome relatedness indexes revealed that the average nucleotide identity and digital DNA–DNA hybridization values between For3^T and its closest phylogenomic relative ( S. atroolivaceus NRRL ISP-5137^T) were 88.39 and 39.2 %, respectively. The G+C content of the genome was 71.4 mol% and its size was 7.96 Mb with 7492 protein-coding genes. Strain For3^T harboured complete metabolic pathways absent in the closest relative strains such as cellulose biosynthesis, glycogen degradation I, glucosylglycerate biosynthesis I. Anteiso-C_15:0, iso-C_15:0, anteiso-C_17:0 and MK-9(H4)/MK-9(H6) were the predominant cellular fatty acids and respiratory quinones, respectively. Phenotypic and genomic data supported the assignment of strain For3^T to a novel species Streptomyces silvae sp. nov., within the genus Streptomyces , for which the type strain is For3^T (=CIP 111908^T=LMG 32186^T).

Actinobacteria are Gram-positive bacteria characterized by a genome with a high G+C ratio. They are numerous and constitute a widely distributed group of soil microbes, representing 10–50% of the soil microflora community. They are important producers of diversified secondary metabolites with antifungal and antibacterial activities [1].

Among this phylum, Streptomyces is the most famous and well-described genus. Streptomyces members are Gram-stain-positive, aerobic and heterotrophic bacteria with aerial mycelia [2]. Streptomyces have a predominant presence of saturated iso and anteiso fatty acids as major cellular fatty acids [3]. The genus Streptomyces , which is very widespread in soils, is a major player in the degradation of organic matter and lignocellulose [4]. It has a large enzymatic arsenal encoding carbohydrate esterases, polysaccharide lyases, glycoside hydrolases and enzymes with auxiliary activities [5, 6] and it can produce approximately around 7600 bioactive compounds [7]. In this study, strain For3^T isolated from forest soil was subjected to a polyphasic taxonomy approach that relied on genomic, chemotaxonomic and physiological data.

Forest soil was sampled in Champenoux, France (48.44° N, 6.21° E) on 13 May 2019 in order to study the actinobacterial strains present in that soil. One gram of the soil sample was placed in 50 ml International Streptomyces Project (ISP) 2 [8] liquid medium at 30 °C for 1 week at pH 7 in order to isolate actinobacteria micro-organisms. After 1 week enrichment, dilution series up to 10⁻⁶ were made; 100 µl of each dilution was plated on ISP 2 agar medium and incubated for 10 days at 30 °C. Among all the isolates after 10 days, one isolate, named For3^T, was then purified by repeated streaking on the same ISP 2 medium and isolated for further study.

The almost-complete 16S rRNA gene sequence of strain For3^T was extracted from the genome sequence and represented a sequence of 1448 nucleotides long. It was deposited in GenBank/EMBL/DDBJ under the accession number MW479423.

Strain For3^T was most closely related to Streptomyces pratensis ATCC 33331^T, Streptomyces anulatus ATCC 27416^T, Streptomyces setonii NRRL ISP-5322^T and Kitasatospora papulosa NRRL B-16504^T[9] with 99.93% 16S rRNA gene sequence similarity. High levels of 16S rRNA gene sequence similarity were previously reported within the genus Streptomyces [10, 11], demonstrating that it is difficult to differentiate Streptomyces species by 16S rRNA gene sequences.

Phylogenetic analysis based on the 16S rRNA gene sequences of strain For3^T and its closest species (determined by blastn) was performed after alignment of sequences using the ClustalW program [12]. Neighbour-joining (Fig. 1) and maximum-likehood phylogenetic trees were built using mega7 software (Fig. S1, available in the online version of this article) [13]. The stability of the grouping was estimated by bootstrap analysis (1000 replicates). The results showed that strain For3^T represents a new branch in both neighbour-joining and phylogenetic trees. Strain For3^T fell within the genus Streptomyces and was closely related to S. pratensis ATCC 33331^T, S. anulatus ATCC 27416^T, S. setonii NRRL ISP-5322^T and K. papulosa NRRL B-16504^T with whom it formed a subgroup.

Fig. 1.

Phylogenetic tree of type strains closely related to strain For3^T (in bold) based on 16S rRNA gene sequences. The evolutionary history was inferred by mega 7.0 [34] using the neighbour-joining method [35]. There were a total of 1315 positions in the final dataset. Bar represents 0.002 substitutions per nucleotide position. Strain For3^T is highlighted in bold.

Genomic DNA was extracted using PureLink Genomic DNA Mini Kit (Invitrogen) according to manufacturer’s instructions. DNA was quantified using the ND-1000 spectrophotometer (NanoDrop Technologies). DNA quality was controlled after electrophoresis on a 0.8% (w/v) agarose gel made in Tris–acetate–EDTA buffer. Genomic DNA was sequenced using a NovaSeq system (Illumina) and was performed by Novogene (Cambridge, UK). The obtained genome sequence was annotated by using the rast server (Rapid Annotation using Subsystem Technology; https://rast.nmpdr.org/) and deposited at DDBJ/ENA/GenBank under the accession number JAERUC000000000. Comparisons between the For3^T genome and those of its closest relatives available in GenBank are presented in Table 1. The genome size of strain For3^T was 7.96 Mbp, slightly smaller than the average genome sizes (8–9 Mbp) reported for the genus Streptomyces [14]. The DNA G+C content of strain For3^T was 71.4 mol%, is near the average of 70 mol% reported for the genus Streptomyces [15].

Table 1.

Genome size, DNA G+C content, digital DNA–DNA relatedness and average nucleotide identity of strain For3^T and closely related type strains of the genus Streptomyces (with a 16S rRNA similarity above 98.65 %)

Strain	Size (bp)	DNA G+C content (mol%)	DNA–DNA relatedness (%)	Average nucleotide identity (%)
Streptomyces sp. For3T	8072 519	71.4
Streptomyces atroolivaceus NRRL ISP-5137T	8 216 207	70.7	39.20	88.39
Streptomyces mutomycini NRRL B-65393T	7 070 041	70.6	39.20	88.85
Streptomyces pratensis ATCC 33331T	7 656 104	71.0	37.2	87.90
Kitasatospora papulosa NRRL B-16504T	7 568 795	71.0	37.10	88.05
Streptomyces clavifer Root1319T	6 744 287	71.3	32.60	86.22
Streptomyces halstedii NRRL ISP-5068T	7 740 838	71.9	30.40	84.66
Streptomyces nitrosporeus ATCC 12769T	7 581 562	72.2	29.30	84.30
Streptomyces setonii NRRL ISP-5322T	7 819 863	71.7	27.40	82.05
Streptomyces brevispora BK160T	7 540224	70.3	27.40	82.18
Streptomyces atratus Ncost-T10-10dT	9 361 102	69.6	27.00	80.94
Streptomyces anulatus ATCC 27416T	9 057 348	71.4	27.00	81.46
Streptomyces peucetius JCM 9920T	8 179 821	72.5	27.00	81.93
Streptomyces cavourensis DSM 41795T	7 670 394	72.1	27.00	82.19
Streptomyces bacilliaris ATCC 15855T	7 888 441	72.0	27.00	82.22
Streptomyces griseus KCTC 9080T	8 630 192	72.2	26.80	81.78
Streptomyces cyaneofuscatus NRRL B-2570T	7 899 007	71.6	26.80	82.11
Streptomyces globisporus NBRC 12867T	8 120 903	71.4	26.70	81.82
Streptomyces parvus NBRC 3388T	8 340 747	71.7	26.60	81.52
Streptomyces badius EN27T	7 550 241	71.6	26.60	82.02
Streptomyces lunaelactis MM109T	8 570 191	69.8	23.90	78.73
Streptomyces michiganensis NBRC 12797T	8 656 216	71.2	23.60	77.88
Streptomyces xantholiticus NBRC 13354T	8 023 114	70.6	23.10	78.27
Streptomyces exfoliatus NRRL B-2924T	7 874 957	71.9	22.80	78.16

The genome blast distance was calculated using the annotation platform system provided by DSMZ (https://tygs.dsmz.de/). The phylogenomic tree obtained (Fig. 2) showed that strain For3^T is individualized in a new branch. Based on that phylogenomic comparison, its closest strain is S. atroolivaceus NRRL ISP-5137^T with an average branch support of 84.9%. Moreover, based on the genomes available, an automated multi-locus species tree was realized using the web server at https://automlst.ziemertlab.com/ [16]. Based on 89 housekeeping core genes (listed in the Table S1), the phylogenomic tree obtained showed that our strain is closely related to S. atroolivaceus NRRL ISP-5137^T (Fig. S2). The pairwise average nucleotide identity (ANI) values were determined by using an ANI calculator (http://jspecies.ribohost.com/jspeciesws/#home) [17]. The ANIb values between For3^T and its closest relatives S. mutomycini NRRL B-65393^T, S. atroolivaceus NRRL ISP-5137^T (the closest phylogenomic strain based on AutoMLST and TYGS), S. pratensis ATCC 33331^T and K. papulosa NRRL B-16504^T were 88.85, 88.39, 87.9 and 88.05% respectively, well below the cut-off value of 95–96% proposed for species delineation [18] (Table 1).

Fig. 2.

Tree inferred with FastME 2.1.6.1 [36] from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d₅. The tree was rooted at the midpoint [37].

Digital DNA–DNA hybridization (dDDH) or DNA–DNA relatedness values were calculated between strain For3^T and the type strains of closest species using the Genome-to-Genome Distance Calculator [19]. The dDDH values between strain For3^T and S. atroolivaceus NRRL ISP-5137^T and S. mutomycini NRRL B-65393^T reached 39.2% (the highest value obtained), lower than the 70% species cut-off value [19, 20] (Table 1). For S. pratensis ATCC 33331^T and K. papulosa NRRL B-16504^T, the ANI values were 37.2 and 37.1% respectively. The tetra method [21] was applied and supported this result with a tetra index of 0.994, below the species delimitation cut-off (>0.999) (data not shown).

Genomic comparisons between strain For3^T and its closest relative strain S. atroolivaceus NRRL ISP-5137^T [22] were performed. Strain For3^T harboured several genes coding enzymes such as 3-hydroxyacyl-[acyl-carrier-protein] dehydratase, 4-carboxy-4-hydroxy-2 oxoadipate aldolase, 4-oxalomesaconate hydratase, clavaldehyde dehydrogenase, creatinine amidohydrolase and threonyl-tRNA synthetase-related protein, unlike the other analysed genomes. Those enzymes could not be related to any specific metabolic pathway. Strain For3^T had 61 genes that were not detected in S. pratensis ATCC 33331^T such as genes encoding for 3-carboxy-cis-muconate cycloisomerase and protocatechuate-3,4-dioxygenase. Strain For3^T had 91 genes that were not detected in K. papulosa NRRL B-16504^T such as several genes in the CRISPR system encoding for CRISPR-associated helicase Cas2, Cas3, Cse3 and Cse4. Strain For3^T had 98 genes that were not detected in S. mutomycini NRRL B-65393^T such as several genes in the arginine and ornithine degradation metabolic pathway such as arginine decarboxylase, ornithine decarboxylase and isochorismatase.

Growth tests at various temperatures, and at different pH and NaCl concentrations, were performed using ISP 2 agar as culture medium. Growth of strain For3^T occurred at 15–30 °C (optimum, 25 °C), at pH 6.0–9.0 (pH 7.0) and in the presence up to 5% w/v NaCl (0% w/v NaCl). Compared to S. pratensis ATCC 33331^T [23], which is the closest strain with physiologic data available, strain For3^T could be distinguished only by a more restricted temperature range growth while the rest of the physiological parameters were the same. Indeed, the growth range of S. pratensis ATCC 33331^T was between 15 and 37 °C. K. papulosa NRRL B-16504^T grew at 15–30 °C (optimum, 25 °C), at pH 6.0–9.0 (pH 7.0) and in the presence up to 5% w/v NaCl (0% w/v NaCl), which are the same values obtained for For3^T. The optimum physiological parameters (pH, temperature) were also close compared to the others strains, especially S. atroolivaceus NRRL ISP-5137^T and S. mutomycini NRRL B-65393^T. Strain For3^T was aerobic, Gram-stain-positive and catalase-positive. Colonies were greenish-white with rough surfaces when the strain was cultivated on ISP 2 medium. Green diffusible pigment was produced on that medium.

The assimilation of carbon sources by strain For3^T was tested using Biolog MicroPlates [24]. Compared to a closest phylogenomic strain S. atroolivaceus NRRL ISP-5137^T [25, 26], strain For3^T was unable to degrade d-glucose and sucrose. Strain For3^T was able to degrade d-fructose, d-mannitol, raffinose and rhamnose, like S. atroolivaceus NRRL ISP-5137^T. Compared to K. papulosa NRRL B-16504^T, strain For3^T was unable to degrade d-glucose; however, strain For3^T was able to utilize d-fructose, d-mannitol, raffinose and rhamnose, whereas K. papulosa NRRL B-16504^T was not. Compared to S. pratensis ATCC 33331^T, both strains were able to degrade fructose, d-mannitol, raffinose and rhamnose; For3^T did not degrade d-glucose in contrast to S. pratensis ATCC 33331^T.

For chemotaxonomic affiliation: (1) the fatty acid methyl esters were obtained from 40 mg fresh scraped colonies from Petri dishes by saponification, methylation and extraction using minor modifications of the method of [27] and [28]; (2) the respiratory quinones were first extracted from 100 mg freeze-dried cell material using methanol–hexane extraction and then separated by using the hexane method described in [29, 30]; (3) the polar lipids were extracted from 200 mg freeze-dried cell material using a choloroform–methanol–0.3% aqueous NaCl mixture; polar lipids were separated by two dimensional silica gel thin-layer chromatography. The first direction was developed in chloroform–methanol–water and the second in chloroform–methanol–acetic acid–water. All analyses relating to fatty acids (www.dsmz.de/services/services-microorganisms/identification/analysis-of-cellular-fatty-acids.html), respiratory quinones (ww.dsmz.de/services/services-microorganisms/identification/analysis-of-respiratory-quinones.html) and polar lipids (www.dsmz.de/services/microorganisms/biochemical-analysis/polar-lipids) were carried out by DSMZ (Braunschweig, Germany). The detection of diamino and mycolic acids in the strain were performed according to standard procedures [31, 32].

For3^T contained MK-9(H6) (60.2 %), MK-9(H4) (29.4 %), MK-9(H2) (3.2 %) and MK-9(H8) (7.2 %) as major respiratory quinones. For3^T had a respiratory quinone content very similar to those of K. papulosa NRRL B-16504^T and S. lunaelactis MM109^T with the presence of MK-9(H2), MK-9(H4), MK-9(H6) and MK-9(H8). Only S. lunaelactis MM109^T had several quinones (MK-9 and MK-10). However, differences in the relative abundance of MK-9(H2), MK-9(H4), MK-9(H6) (always the most abundant) and MK-9(H8) were detected. S. pratensis ATCC 33331^T had only MK-9(H6) and MK-9(H8).

The dominant fatty acids of strain For3^T were anteiso-C_15:0 (32.97 %), iso-C_15:0 (15.37%) and anteiso-C_17:0 (13.83 %). This cellular fatty acid profile was consistent with those of members of the genus Streptomyces such as S. pratensis ATCC 33331^T, S. lunaelactis MM109^T and S. brevispora BK160^T_.

Strain For3^T had lipid, glycolipid, aminolipid, phosphatidylethanolamine and diphosphatidylglycerol as polar lipids (Fig. S3). Only this strain had such diversity in term of polar lipid profile: indeed, for the others strains, S. lunaelactis MM109^T had lipid, phosphatidylethanolamine and diphosphatidylglycerol, whereas K. papulosa NRRL B-16504^T had only phosphatidylethanolamine. Concerning the detection of diamino acid in the whole-organism hydrolysate, ll-diaminopimelic acid (A2pm) was found, confirming that the strain belongs to the genus Streptomyces [33]. Strain For3^T lacked mycolic acid.

In conclusion, the respiratory quinone, fatty acid and polar lipid profiles, as well as 16S rRNA gene sequencing results, showed unambiguously that strain For3^T is affiliated to species of the genus Streptomyces . On the basis of the phenotypic differences observed and the results of genomic studies, strain For3^T can be clearly distinguished from the most closely related species of the genus Streptomyces . Therefore, we propose a novel species within the genus Streptomyces , with the name Streptomyces silvae sp. nov. and with strain For3^T as type strain.

Description of Streptomyces silvae sp. nov.

Streptomyces silvae (sil'vae. L. gen. n. silvae of a forest).

Aerobic, Gram-stain positive, catalase-positive actinobacterium (0.5×1.5 µm) (Fig. S4). Green diffusible pigment is formed on ISP 2 medium and the strain differentiates into spiral chains of spores with rough surfaces on sporulation medium. Growth occurs at 15–30 °C (optimum, 25 °C), at pH 6.0–9.0 (pH 7.0) and in the presence up to 5% w/v NaCl (0% w/v NaCl). Able to degrade d-arabitol, d-aspartic acid, cellobiose, dextrin, d-fructose, d-fructose-6-PO₄, d-fucose, d-galactose, d-gluconic acid, d-glucose-6-PO₄, d-glucuronic-acid, d-lactic acid, methyl ester, d-malic acid, maltose, d-mannitol, d-mannose, melibiose, raffinose, d-salicin, d-sorbitol, formic acid, gentiobiose, glucuronamide, l-alanine, l-arginine, l-aspartic acid, l-fucose, l-galactonic acid, lactone, l-glutamic acid, lithium, chloride, l-lactic acid, l-rhamnose, l-serine, mucic acid, myo-inositol, N-acetyl neuraminic acid, N-acetyl-d-galactosamine, pectin, p-hydroxy-phenylacetic acid, potassium tellurite, rifamycin SV, sodium bromate, sodium butyrate, stachyose, α-hydroxy-butyric acid and α-keto-butyric acid. Strain For3^T is not able to degrade 3-methyl-glucose, acetic acid, acetoacetic acid, aztreonam, bromo-succinic acid, citric acid, d-galacturonic acid, d-saccharic acid, d-serine, trehalose, turanose, fusidic acid, gelatin, glycerol, glycyl-l-proline, guanidine HCl, inosine, l-histidine, lincomycin, l-malic acid, l-pyroglutamic acid, methyl pyruvate, minocycline, N-acetyl-d-glucosamine, N-acetyl-β-d-mannosamine, nalidixic acid, niaproof 4, propionic acid, quinic acid, sucrose, troleandomycin, Tween 40, vancomycin, α-d-glucose, lactose, α-keto-glutaric acid, β-hydroxy-d, l-butyric acid, methyl β-d-glucoside and γ-amino-butyric acid. Lipid, glycolipid, aminolipid, phosphatidylethanolamine and diphosphatidylglycerol are present as polar lipids. Anteiso-C_15:0, iso-C_15:0 and anteiso-C_17:0 are the most abundant fatty acids. MK-9(H4) and MK-9(H6) are the predominant quinones.

The type strain, For3^T (=CIP 111908^T=LMG 32186^T), was isolated from a forest soil sample collected near Champenoux, France.